In this project, we are focusing primarily on determining the mechanisms of morphogenesis of salivary glands and other organs. We are addressing the following major questions: 1. How do embryonic salivary glands and other branched organs generate their characteristic branched architectures during the process of branching morphogenesis? Specifically, how is the formation of clefts, buds, and ducts mediated and coordinated at molecular and biophysical levels? How can we facilitate bioengineering for organ replacement - particularly of salivary glands - by understanding branching morphogenesis and by promoting specific steps of this process? 2. What are the contributions of selective, local regulation of organ-specific gene expression, extracellular matrix, integrins, signal transduction, and cell migration to organ branching morphogenesis and organ specification? Branching morphogenesis of developing organs requires coordinated but still relatively poorly understood changes in gene expression, epithelial cell-cell adhesion, and cell motility. To obtain sensitive and specific characterization of the expression localization patterns of individual genes, methods for single-molecule FISH (fluorescence in situ hybridization) at low cost were developed, along with methods to quantify mRNA levels in each cell. Single-cell and bulk RNA transcriptome analyses are being performed on embryonic submandibular versus parotid salivary glands to characterize their molecular identities during early bud initiation. Salivary gland mesenchymal cells were considerably more heterogeneous by clustering analysis than the epithelial cells. These analyses further revealed the presence of well-defined clusters of mesenchymal cells specific to each gland at even the one-bud stage of development. At this very early developmental stage, a muscle-related gene expression cluster was prominent in the parotid, but not in the submandibular gland. These findings suggest that distinct transcriptional identities emerge early in development of these two types of salivary gland. Our previous studies identified highly dynamic interactions of cells with basement membranes during organ development. For example, epithelial cells translocate actively along basement membranes, but they also produce numerous microscopic basement membrane perforations through which they protrude blebs and longer dynamic cell extensions. In order to define more precisely the relationships of peripheral salivary gland epithelial cells to the basement membrane, studies are underway to track the movements of both individual epithelial cells and basement membranes by time-lapse multiphoton confocal microscopy. The relative contributions of cell-basement membrane interactions, epithelial cell proliferation and motility, and E-cadherin mediated cell-cell interactions are being explored in mechanistic depth. The protein Btbd7 has been implicated in branching morphogenesis, but it has not been characterized in depth at a molecular level. We are attempting to develop a monoclonal antibody against this protein to characterize its localization patterns and interactions in depth. For this purpose, an ultra-sensitive western blotting methodology was developed, which can detect antigen-binding activity in nanoliter quantities of serum with low assay background. It can be used to screen hybridoma supernatants and to characterize other antibodies using very limited amounts of sample. These studies are beginning to elucidate the complex regulatory systems important for the cell and tissue dynamics involved in craniofacial organ development, particularly of salivary glands. Understanding these underlying morphogenetic mechanisms should promote more effective tissue engineering for restoration of damaged organ function.